Biology of Blood and Marrow Transplantation 12:160-171 (2006) ᮊ 2006 American Society for Blood and Marrow Transplantation 1083-8791/06/1202-0005$32.00/0 doi:10.1016/j.bbmt.2005.10.027 Impaired Allogeneic Activation and T-helper Differentiation of Human Cord Blood Naive CD4 T Cells Li Chen, Aileen C Cohen, David B Lewis Department of Pediatrics and the Immunology Program, Stanford University School of Medicine, Stanford, California Correspondence and reprint requests: D B Lewis, MD, Division of Immunology and Transplantation Biology, Department of Pediatrics, Stanford University School of Medicine, CCSR Bldg, Room 2115b, 269 Campus Dr, Stanford, CA 94305 (e-mail: dblewis@stanford.edu) Received June 25, 2005; accepted October 22, 2005 ABSTRACT CD4 T cells, particularly those of the T-helper (Th1) subset, are important effectors in alloimmune diseases, such as graft-versus-host disease, and in controlling infections with intracellular pathogens Thus, it is plausible that impaired neonatal CD4 T-cell immunity might contribute to the low incidence of acute graft-versus-host disease after allogeneic transplantation of hematopoietic stem cells using cord blood (CB) compared with adult sources of hematopoietic stem cells In support of this hypothesis, we found that CB naive CD4 T cells had reduced activation and impaired early Th1 differentiation compared with adult peripheral blood naive CD4 T cells after stimulation by allogeneic dendritic cells derived from adult monocytes Early Th1 polarization was dependent on interleukin-12 and CD154, and CB CD4 T cell/dendritic cell co-cultures had impaired expression of both proteins CB naive CD4 T cells had low basal levels of signal transduction and activation of transcription messenger RNA and protein, and, after alloantigen stimulation, reduced interleukin-12-induced signal transduction and activation of transcription tyrosine phosphorylation, compared with adult peripheral blood naive T cells Lastly, FoxP3 protein expression, a marker for regulatory CD25high CD4 T cells, was lower for naive CD4 T cells of CB compared with those of adult peripheral blood, which argued against increased T-regulatory activity as a mechanism for the decreased Th1 differentiation of CB CD4 T cells Together, these intrinsic limitations in T-cell activation and Th1 differentiation may impair the ability of T cells in CB and the neonate to respond to allogeneic or infectious challenges © 2006 American Society for Blood and Marrow Transplantation KEY WORDS Cord blood ● Naive CD4 T cell ● Dendritic cell INTRODUCTION Acute graft-versus-host disease (GVHD), in which immunocompetent transplanted donor T cells recognize alloantigens presented by host cells [1,2], remains a major barrier to successful transplantation of allogeneic hematopoietic stem cells After their infusion, allogeneic donor T cells predominantly migrate to lymphoid tissues where they become activated by interacting with host or donor antigen-presenting cells [3], particularly the dendritic cell (DC) [4] Recent studies suggest that CD4 antigenically naive rather Li Chen and Aileen Cleary Cohen contributed equally to this report 160 ● Allogeneic activation ● T-helper cells than memory T cells are mainly activated in the recipient and are critical in initiating acute GVHD in vivo [5-7] Activated CD4 T cells then travel to nonlymphoid tissues, such as the skin, gastrointestinal tract, and liver, followed by CD8 T cells, where they cause tissue damage [7] as effector CD4 and CD8 T cells producing proinflammatory cytokines, such as interferon (IFN)-␥ and tumor necrosis factor-␣, and cytotoxins [8] The importance of effector CD4 T cells of the T-helper (Th1) type, which produce IFN-␥ but not T-helper cytokines, in the immunopathogenesis of acute GVHD, especially for intestinal pathology [9], is supported by a number of animal studies [1] Impaired Neonatal CD4 T-Cell Allogeneic Activation Allogeneic hematopoietic stem cell transplantation using umbilical vein cord blood (CB) is associated with a decreased incidence and severity of acute GVHD compared with transplantation using adult peripheral blood (APB) or bone marrow cells [10,11] Because CB contains a substantially higher concentration of naive T cells than either APB [12] or bone marrow [13], these findings suggest that transplanted CB naive T cells have a reduced capacity to mediate alloimmune responses in vivo However, the mechanisms that are responsible for decreased GVHD after allogeneic CB transplantation remain unclear Th1 effector cells are important for the control of intracellular microbes, particularly bacteria [14] and contributing to the immunopathology of acute GVHD [1] The activation and differentiation of naive CD4 T cells into Th1 effectors is initiated by engagement of ␣T-cell receptor (TCR) with antigenic peptide/major histocompatibility complex (MHC) class II complexes [15] DCs express high levels of MHC class II and costimulatory ligands, such as CD80 and CD86, and are essential for activating naive CD4 T cells in response to foreign antigens [16] and MHC alloantigens in vivo [4] DCs promote Th1 differentiation by secreting cytokines, such as interleukin (IL)-12, especially after engagement of their CD40 molecule by CD154 (CD40-ligand) on activated naive CD4 T cells [17] Activated naive CD4 T cells also secrete IL-2 and acquire high-affinity receptors for IL-2 and IL12, which results in IL-2- and IL-12-dependent T-cell proliferation [15,17] and facilitation of IFN-␥ gene transcription [18] The biologic effects of IL-12 are mainly dependent on tyrosine phosphorylation of signal transducer and activator of transcription (STAT4) [19] that occurs after IL-12 receptor engagement by its ligand Key STAT4-dependent events include increased CD4 T-cell expression of IFN-␥ [20], which, in turn, may enhance CD4 T-cell expression of T-bet, a transcription factor that strongly favors Th1 and inhibits T-helper development [21] IFN-␥ produced early after naive CD4 T-cell activation may also favor Th1 differentiation by helping to maintain functional IL-12 receptors on the T-cell surface [20] Regulatory CD4 T cells (Tregs) comprise a wellcharacterized population of cells that express high levels of CD25 and play an important role in the negative control of inflammation and in the maintenance of tolerance to self [22,23] Both human and murine Tregs express high levels of the forkhead family of transcription factors member FoxP3, whose expression is essential for programming Treg cell development and function [24,25] CB from term pregnancies has been shown to have equivalent numbers of CD4 T cells that are CD25high as APB [26] These CD25high CB T cells are able to suppress antigen-specific T-cell BB&MT responses in vitro [27,28], although to a lesser extent than CD25high CD4 T cells of APB [29] The possibility that decreased acute GVHD seen in CB transplantations might be caused by impaired Th1 differentiation of CB CD4 T cells is currently not resolved There is evidence that CD154 induction may be reduced on CB-derived T cells versus those derived from adult blood sources [30-33] However, others have demonstrated equivalent expression of CD154 after stimulation [34-36] Although some studies show equivalent levels of IFN-␥ by CB and APB T cells after stimulation [34,35], others have shown impaired IFN-␥ production [37,38], depending on the conditions used for stimulation [39] Importantly, none of the above studies analyze the early events of Th1 differentiation using purified CD4 naive T cells and allogeneic stimulation with mature DCs, the interaction thought to initiate acute GVHD [1] We show here that naive CD4 T cells of CB compared with those of APB have impaired early activation, IFN-␥ expression, and IL-12 responsiveness after allogeneic stimulation with DCs, and an impaired ability to induce IL-12 production by DCs Thus, multiple intrinsic limitations in CB CD4 T-cell activation, which also limit DC function, impair the differentiation of this CD4 T-cell population into effector Th1 cells, and these limitations account, in part, for the decreased risk of acute GVHD after CB transplantation We further found that FoxP3 expression, which is strongly associated with Treg cell function, is decreased in CB naive CD4 T cells that are CD25high, suggesting that these limitations in Th1 differentiation are an intrinsic property of naive CB CD4 T cells rather than a result of an increase in the proportion of naive cells that are regulatory T cells MATERIALS AND METHODS CD4 T-Cell Purification Naive (CD45RAhighCD45R0low) CD4 T cells were purified by negative selection from APB or CB mononuclear cells of full-term neonates prepared by FicollHypaque density gradient centrifugation of anticoagulated blood The mononuclear cells were incubated with RosetteSep Cocktail for CD4 T cells (Stemcell Technologies, Vancouver, British Columbia, Canada), followed by incubation with paramagnetic beads coated with monoclonal antibodies (mAbs) against non-T-cell markers, CD8-␣, and CD45R0 (Miltenyi, Auburn, Calif), and application to a magnetic-activated cell sorter Final purity as assessed by flow cytometry was routinely more than 95% Monocyte-Derived DCs Monocytes were isolated from APB mononuclear cells using CD14 mAb-coated paramagnetic beads 161 L Chen et al (Miltenyi) and positive selection using the magneticactivated cell sorter Monocytes were cultured (5.0 ϫ 105/mL) for days in complete RPMI-1640 medium [40] with 10% heat-inactivated human AB serum plus recombinant human granulocyte macrophage-colonystimulating factor (25 ng/mL) (Immunex Corp, Seattle, Wash) and recombinant IL-4 (20 ng/mL) (Peprotech, Rocky Hill, NJ) Recombinant human tumor necrosis factor-␣ (10 ng/mL) (Biosource, Camarillo, Calif) was added after day 7, and mature monocyte-derived DCs were harvested on day and cryopreserved in 7% (vol/ vol) dimethylsulfoxide (Sigma Chemical, St Louis, Mo) in heat-inactivated human AB serum Unless otherwise indicated, equal numbers of DCs from unrelated donors were pooled together for allogeneic stimulation Allogeneic Stimulation of Naive CD4 T Cells Equal numbers (2 ϫ 105-1 ϫ 106 at 1.5 ϫ 106/mL) of adult or CB-derived naive CD4 T cells were stimulated with allogeneic DCs (1.5 ϫ 105/mL) in 200 L of complete RPMI-1640 medium in 96-well flat-bottom plates for 24 to 72 hours For intracellular cytokine staining, brefeldin A (10 g/mL) (Sigma Chemical) was added for the last hours of a 48-hour stimulation kit, R and D Systems) The lower limit of sensitivity for each of the cytokine enzyme-linked immunosorbent assays were as follows: IFN-␥, 1.0 pg/mL; IL-2, 1.0 pg/mL; IL-4, 2.0 pg/mL; IL-10, 2.0 pg/mL; and IL-12, 0.5 pg/mL Cell Staining and Flow Cytometric Analysis Staining for cell surface proteins and intracellular cytokine staining, poststaining fixation, and flow cytometric analysis were performed as previously described [40,41] The number of cells analyzed per condition by flow cytometry was no less than 10,000 naive CD4 T cells, and was often between 20,000 to 50,000 events, depending on the experiment FoxP3 staining was accomplished using anti-FoxP3 antibodies directly conjugated to FITC versus isotype controls (both from eBiosciences) These were used in the intracellular staining assay following a protocol provided by the manufacturer Real-Time Reverse-Transcriptase Polymerase Chain Reaction Analysis Mouse-antihuman biotinylated CD154 (clone 2431) (Ancell, Bayport, Minn); phycoerythrin (PE)-Cy5CD4 (clone S3.5) (Caltag, Burlingame, Calif); PECD69 (clone CH4, Caltag); fluorescein isothiocyanate (FITC)-CD45RA (clone L48) (BD Biosciences, Mountain View, Calif); allophycocyanin-CD45RO (clone UCHL1) (BD Biosciences); PE-CD25 (clone CD253G10) (Caltag); and FITC-IFN-␥ (clone 25723.11) (BD Biosciences) mAbs were used for flow cytometric analysis and cell isolation Appropriate biotinylated or fluorochrome-conjugated mouse isotype control mAbs (Caltag) were used as negative controls Unconjugated mouse antihuman CD154 (clone, 5C8, IgG2a) and IL-12 (clone 24910.1, IgG1) (R and D Systems, Minneapolis, Minn) mAbs or equivalent concentrations of sodium azide-free murine IgG1 and IgG2a isotype controls (Sigma Chemical) were used for neutralization experiments Rabbit antihuman STAT4 and phosphotyrosine (pY)-STAT4 antisera, and preimmune sera were purchased from Zymed, South San Francisco, Calif FoxP3 antibody and isotype control were purchased from eBioscience, San Diego, Calif Total RNA was isolated, converted to complementary DNA, and polymerase chain reaction amplified in triplicate using an ABI Prism 5700 instrument (Applied Biosystems, Foster City, CA) and SYBR green dye (Applied Biosystems, Foster City, CA) as previously described [40] Oligonucleotide primer pairs used for amplification were human STAT4 [nucleotide (nt)1387F 5=-TGCCTCTATGGCCTGACCAT-3= and nt1438R 5=-TCACCACAGGCAATGAGCTG-3=] and previously reported [40] 18S ribosomal RNA primers The cycle threshold (Ct) value, the calculated polymerase chain reaction cycle at which products first became detectable, was determined using software (ABI) Ct values for 18S ribosomal RNA, which assessed the effectiveness of reverse transcription and polymerase chain reaction amplification, were used for normalization of the STAT4 transcript Ct values Levels of STAT4 messenger RNA (mRNA) relative to those found in freshly isolated naive CB CD4 T cells were calculated from Ct values using the formula 2Ϫ⌬Ct Total RNA from CD69hi CD4 T cells was isolated after 24 hours of stimulation with allogeneic DCs by incubating cultured cells with PE-conjugated CD69 mAb, and positively selecting CD69hi cells using PE-specific paramagnetic microbeads (Miltenyi) and a magnetic-activated cell sorter The final CD69hi cell fraction contained less than 0.1% DCs based on light microscopic cellular morphology Cytokine Assays STAT4 Flow Cytometric Assays Cell culture supernatants were analyzed for cytokine content using enzyme-linked immunoassay kits for IFN-␥, IL-2, IL-4, IL-10 (OptEIA, BD Biosciences), and IL-12 (Quantikine HS immunoassay Intracellular total STAT4 staining was performed as previously described [42] For pY-STAT4 staining, naive CD4 T cells were allogeneically stimulated for 48 hours and then incubated with 20 ng/mL of re- Primary Antibodies 162 Impaired Neonatal CD4 T-Cell Allogeneic Activation combinant IL-12 (Genetics Institute, Boston, Mass) for 20 minutes at 37°C Cells were treated with fixation reagent A (Fix and Perm, Caltag) for to minutes at room temperature, followed by the addition of an equal volume of ice-cold methanol for 10 minutes Cells were washed, permeabilized with reagent B (Caltag), stained at room temperature with primary STAT4 antibodies or preimmune rabbit sera for 30 minutes, followed by incubation with FITCconjugated goat antirabbit IgG (Caltag) for 20 minutes, and then immediately analyzed by flow cytometry Statistics Data are presented as mean Ϯ SEM P values for differences between means were calculated using the 2-tailed, unpaired Student t test RESULTS Reduced Naive CB CD4 T-Cell Expression of CD69 and CD154 after Stimulation by Allogeneic DCs To determine if CB and APB naive CD4 T cells differed in their ability to respond to optimal antigenic stimulation, these ells were co-cultured with monocyte-derived DCs from unrelated adult blood donors DCs were selected as allogeneic stimulators because of their physiologic relevance as antigen-presenting cells for naive CD4 T-cell activation in vivo [16], including in the context of GVHD [4], and their potency as activators, which enhanced our ability to analyze T-cell responses using flow cytometry Naive CD4 T cells co-cultured individually with different allogeneic DC lines were first analyzed for their expression of CD69 and CD154, which are induced early after activation [43,44] Each DC line induced a substantial proportion of APB naive CD4 T cells to express CD69 (Figure 1A) Pooling of the DC lines from unrelated individuals consistently induced greater amounts of CD69 than any of the individual lines, consistent with the pool providing a greater number of MHC class II alloantigenic determinants CB naive CD4 T cells under these conditions had a significantly lower percentage of CD69ϩ cells than APB naive cells (CB and APB CD4 T-cell percentages of 6.60 Ϯ 1.35 and 13.08 Ϯ 2.68, respectively, n ϭ 6, P ϭ 005) The amount of CD69 per cell was similar based on mean Figure Reduced activation of CB naive CD4 T cells by allogeneic DCs CD69 (A) and CD154 (B) expression on APB and CB naive CD4 T cells after incubation with or without allogeneic DCs for 24 hours Cells were analyzed by flow cytometric analysis for CD4, CD45RA, and CD154 or CD69 DC1, DC2, and DC3 represent DC lines derived from unrelated healthy adult blood donors, respectively, and DC1ϩ2ϩ3 indicates pool of these DC lines Events shown are gated for CD45RAϩ lymphocytes, with boxed numbers indicating percentage of positive cells Results shown are representative of at least independent experiments BB&MT 163 L Chen et al Figure Reduced cytokine production by co-cultures of CB naive CD4 T cells with allogeneic DCs A, IFN-␥, IL-2, IL-4, IL-10, and IL-12 content in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA) of naive CD4 T cells from APB (open bars) or CB (closed bars) after 48 hours of co-culture with allogeneic DCs *P Ͻ 05 compared with adult CD4 T-cell co-cultures B, Kinetics of IFN-␥ and IL-2 accumulation in supernatants of ABP (solid line) or CB (dashed line) naive CD4 T-cell co-cultures with allogeneic DCs by ELISA Results are representative of independent experiments fluorescent index (MFI) measurements (CB and APB CD4 T-cell MFI of 237 Ϯ 19 and 282 Ϯ 18, respectively, P ϭ 12) In contrast, CB naive CD4 T cells had a significant reduction in both the percentage of CD154 positive cells (Figure 1B; CB and APB CD4 T-cell values of 2.9 Ϯ 0.4 and 5.9 Ϯ 0.7, respectively, n ϭ 7, P ϭ 001) and the level of CD154 per positive cell (CB and APB CD4 T-cell MFI values of 134 Ϯ 18 and 325 Ϯ 52, P ϭ 002) In subsequent experiments, a pool of DC lines was used to maximize the CD4 T-cell response to alloantigens, and to limit any potential impact of chance matching of MHC class II alleles between stimulator and responder cells 164 Reduced IFN-␥, IL-2, and IL-12 Production by Cultures of CB CD4 T Cells with Allogeneic DCs The cytokine content of supernatants from cocultures of CB or APB naive CD4 T cells with allogeneic DCs was analyzed As expected, IFN-␥, IL-2, IL-4, IL-10, and IL-12 were not detected in supernatants of naive CD4 T cells or DCs cultured alone (Figure 2A) Co-cultures of APB naive CD4 T cells with DCs produced substantially greater amounts of IFN-␥, IL-2, and IL-12 compared with those containing CB CD4 T cells (Figure 2A) Peak levels of IL-2 and IFN-␥ were achieved at 48 and 72 hours of incu- Impaired Neonatal CD4 T-Cell Allogeneic Activation Figure IFN-␥ production by naive CD4 T cells after allogeneic DC stimulation is dependent on IL-12 and CD154 A, APB or CB naive CD4 T cells were incubated with allogeneic DCs for 48 hours in presence or absence of 10 g/mL of neutralizing CD154 or IL-12 p70 mAb or isotype-matched control mAbs Intracellular IFN-␥ accumulation in APB (top) or CB (bottom) naive CD4 T cells is shown Events shown are CD45RAhigh lymphocytes, with boxed numbers indicating percentage and MFI of IFN-␥ϩ cells B, IFN-␥ or IL-12 levels in supernatants of co-cultures of CD4 T cells and DCs AB or CB naive CD4ϩ T cells (3 ϫ 105) were incubated with allogeneic DCs (3 ϫ 104) in presence or absence of anti-CD154 antibody (5c8), or anti-IL-12 or their isotype control antibodies IgG2a, or IgG1, at 10g/mL After 48 hours of culture, supernatants were harvested and measured for IFN-␥ or IL-12 content by enzyme-linked immunosorbent assay *P Ͻ 05 compared with isotype control mAb incubation Results shown are representative of independent experiments bation, respectively, in APB or CB CD4 T-cell cocultures with DCs (Figure 2B) Substantially lower levels of IFN-␥ were also observed in CB CD4 T cell/DC cultures compared with cultures of ABP naive CD4 T cell/DC cultures for at least 120 hours of incubation (Figure 2B) This suggests that the events of T-cell activation required for de novo IL-2 and IFN-␥ mRNA expression by CD4 T cells, e.g., activation of nuclear factor of activated T-cell proteins BB&MT [45], occurred in CB CD4 T cells without a major lag The delayed appearance of IFN-␥ relative to IL-2 that was observed was also consistent with most IFN-␥ expression, but not IL-2 expression, requiring chromatin remodeling linked with cell division [18] Reduced intracellular accumulation of IFN-␥ by CB naive CD4 T cells after co-culture with allogeneic DCs was also evident (Figure 3A), with both the frequency of IFN-␥ϩ cells and the amount of IFN-␥ 165 L Chen et al produced per positive cell, based on MFI measurements, being substantially lower compared with APB naive CD4 T cells No IFN-␥ accumulation by DCs could be detected in these co-cultures (data not shown), although DCs are a potential source of this cytokine [46] We were also unable to detect either IL-2 or IL-12 expression by co-cultured cells using intracellular cytokine staining, most likely because of the relatively low levels of these proteins per cell However, CD4 T cells were likely the major source of IL-2 and DCs the major source of IL-12, based on previous studies by others [15,17] Cultures of CB CD4 T Cells and Allogeneic DCs Produce IL-10 but Not IL-4 The production of IL-4 by either APB or CB CD4 T-cell co-cultures with DCs was low to undetectable (Figure 2A) Thus, there was no evidence for CB CD4 T cells being prone to T-helper polarization after optimal stimulation with mature DCs, although this might still apply to less optimal activation conditions [47] APB and CB CD4 T-cell co-cultures with DCs both produced readily detectable and similar amounts of IL-10 (Figure 2A), which has multiple antagonistic effects on the generation of Th1 immunity [48] As was the case for IL-2 and IL-12, intracellular staining for IL-10 was not detectable so that the relative contribution of DCs and CD4 T cells to IL-10 production could not be determined Role of Decreased CD154 and IL-12 in Limiting CB CD4 Th1 Polarization Previous studies have shown that CD154 expression and IL-12 are critical for the in vivo accumulation of human Th1 memory cells [40,49] In co-cultures of APB naive CD4 T cells with allogeneic DCs, we found that specific neutralization of either CD154 or IL-12 reduced the frequency of CD4 T cells that expressed IFN-␥ intracellularly (Figure 3A) and the level of IFN-␥ protein in cell culture supernatants (Figure 3B) and this occurred to a similar extent with either APB or CB-derived naive CD4 T cells IL-12 levels were also specifically and markedly decreased in cultures containing neutralizing CD154 mAb (Figure Figure Reduced STAT4 expression and IL-12-induced tyrosine phosphorylation of STAT4 by CB naive CD4 T cells A, STAT4 mRNA levels in CB and APB naive CD4 T cells immediately after isolation or in naive CD4 T cells expressing CD69 after 24 hours of alloantigenic stimulation Transcript level for freshly isolated CB naive CD4 T cells was arbitrarily assigned value of 1.0 for comparison Ct values were normalized using 18S ribosomal RNA as internal control *P Ͻ 05 compared with corresponding CB CD4 T-cell subset B, Intracellular STAT4 protein in APB (top) or CB naive (bottom) CD4 T cells, with number in left upper corner indicating percentage of cells staining positive by flow cytometry C, Flow cytometric analysis of intracellular pY-STAT4 expression by allogeneically primed CB and APB naive CD4 T cells after brief treatment with IL-12 Events shown are for CD45RAϩ CD4 T cells, with numbers indicating percentage of positive cells For all panels, one of independent experiments with similar results is shown 166 Impaired Neonatal CD4 T-Cell Allogeneic Activation Figure Decreased FoxP3 expression by naive CD4 T cells of the CD25high subset from CB compared with APB Flow cytometric analysis of naive (CD45RAhigh) CD4 T cells from APB (top) and CB (bottom) are shown A, CD4 versus CD25 surface expression, with percentage CD25Ϫ/low cells (low right quadrant) and percentage of CD25high cells and MFI for CD25 of CD25high population (upper right quadrant) indicated B, Intracellular FoxP3 expression in freshly isolated CD25high naive CD4 T cells, with percentage of positive cells indicated Filled histogram (isotype control) is compared with solid line (FoxP3) C, Intracellular FoxP3 expression in freshly isolated CD25Ϫ/low naive CD4 T cells Filled histogram (isotype control) is compared with solid line (FoxP3), and percentage of positive cells is indicated 3B), indicating that IL-12 production was dependent, in part, on CD154 expression Decreased STAT4 Expression and Activation in Naive CB CD4 T Cells We next determined if decreased expression of STAT4, which plays a critical role in Th1 differentiation [20,21], might contribute to reduced CB CD4 T-cell IFN-␥ production after allogeneic stimulation Freshly purified CB naive CD4 T cells had STAT4 transcript levels that were approximately 20-fold lower than those of APB naive CD4 T cells (Figure 4A) These marked differences in STAT4 mRNA expression were also observed at the protein level (Figure 4B) To determine if STAT4 expression was increased by allogeneic priming, total RNA from CB or APB naive CD4 T cells that were CD69ϩ after coculture with allogeneic DCs was analyzed Although priming consistently increased STAT4 mRNA expression for both CD4 T-cell types relative to their basal levels, primed CB CD4 T cells still had reduced levels of STAT4 transcripts (Figure 4A) and protein (data not shown) compared with APB cells These differences in STAT4 expression were of functional significance, in that allogeneically primed CB naive CD4 T cells had a substantially decreased expression of pYSTAT4 after IL-12 treatment compared with similarly cultured and IL-12-treated APB naive CD4 T cells (Figure 4C) No pY-STAT4 was detectable in cultured CD4 T cells in the absence of IL-12 treatment Reduced FoxP3 Expression in CD25high CB Naive CD4 T Cells CD25high CD4 T cells include a population of Tregs that are known to be important in preventing BB&MT autoimmune disease, in mediating peripheral transplantation tolerance, and in dampening the immune response to endogenous bacterial flora and pathogens [22,23] Thus, it is plausible that these cells may also limit the activation of and cytokine production by naive CD4 T cells in acute GVHD To determine whether an increased proportion of Tregs among naive CD4 T cells in CB might limit their ability to differentiate into Th1 cells, we first examined the percentage of naive CD4 T cells that expressed CD25 in CB versus APB A slightly lower percentage of CB naive CD4 T cells expressed high levels of CD25 versus APB naive CD4 T cells (Figure 5A) Because FoxP3 expression has been strongly correlated to Treg function [24,25], we next examined FoxP3 protein level in APB versus CB naive CD4 T cells Although a substantial fraction of CD25high naive CD4 T cells in both CB and APB expressed FoxP3 protein, this percentage for APB CD25high CD4 naive T cells was almost twice that of the analogous CD25high CD4 naive T-cell population in CB (Figure 5B) Neither APB nor CBderived CD4 T cells that were CD25Ϫ/low expressed FoxP3 protein (Figure 5C) Because FoxP3 expression is thought to tightly correlate with Treg development and function, these data suggest that increased Treg activity within the CB naive CD4 T-cell population does not play a role in limiting activation and differentiation into Th1 cells compared with APB naive CD4 T cells DISCUSSION The transplantation of allogeneic CB is associated with a lower incidence and severity of acute GVHD compared with transplantation using allogeneic pe167 L Chen et al ripheral blood or bone marrow from adults as sources of hematopoietic stem cells [10,11] Recent studies suggest that the naive rather than memory/effector CD4 T cells are an important determinant of acute GVHD [5-7] and, therefore, that naive T cells are important precursors of effector cells producing Th1 cytokines and cytotoxins that are implicated in this disease [1,2] Because CB contains a substantially higher concentration of naive CD4 and CD8 T cells expressing a diverse surface ␣-TCR repertoire [50] than either ABP [12] or bone marrow [13], these findings suggest that the functional capacity of naive T cells in CB to mediate GVHD after transplantation may be limited compared with naive T cells from adult sources In this study, we used allogeneic DCs, a cell type that is highly effective in naive T-cell activation [16] and that is likely to play a central role in acute GVHD [4], to activate highly purified naive CD4 T cells from CB or APB in vitro Our results reveal multiple T-cell intrinsic mechanisms that are likely to limit the acquisition of Th1 effector function in vivo by naive CD4 T cells in CB after their transplantation into an allogeneic recipient The major difference between our study and previous observations that compare CD154 or cytokine expression using APB or CB T cells [34,35,37,38] is the demonstration of impairment in early allostimulation events using mature DC populations that are co-cultured with purified naive CD4 T cells rather than a mixture of naive and memory cells A comparison of purified naive CD4 T cells from APB versus CB is important because memory T cells have a substantially lower activation threshold and mediate rapid effector responses compared with naive T cells [51] We also focused on naive CD4 T cells, because this subset appears to be critical in the initiation of acute GVHD immunopathogenesis [5-7] Another difference from earlier studies using allogeneic stimulation with DCs [34,35] is that we analyzed our cells and culture supernatants for activation-dependent proteins and responsiveness to IL-12 at a shorter interval after stimulation (i.e., 24-120 hours) rather than longer periods In contrast to other studies [34,35], we also did not perform restimulation of alloantigen primed CD4 T cells with pharmacologic stimuli, such as calcium ionophore and phorbol ester Our focus was to determine whether there were differences in allogeneic activation and early Th1 differentiation between naive CD4 T cells from CB versus APB that might have clinical relevance to the early events in the initiation of acute GVHD Despite their diverse ␣-TCR repertoire, CB naive CD4 T cells had a significantly lower percentage of cells expressing CD69 than APB naive CD4 T cells after allogeneic DC stimulation The level of CD69 per cell was similar for both naive CD4 T-cell types In contrast, both the frequency and the amount of 168 CD154 per cell were significantly reduced on CB naive CD4 T cells compared with APB cells These results obtained using a physiologically relevant antigen-presenting cell population for ␣-T-cell stimulation support previous observations in which engagement of the ␣-TCR/CD3 complex using mAbs and bacterial superantigens was used, and which also showed that CB naive CD4 T cells had substantially greater limitations in CD154 than in CD69 expression compared with APB CD4 T cells [33] These differences in the capacity of CB CD4 T cells for the expression of CD69 versus CD154 are consistent with an early bifurcation in the signal transduction pathways that lead to the induction of de novo transcription of the CD69 and CD154 genes after T-cell activation: CD69 gene expression is mainly dependent on phorbol ester-inducible events [52], such as activation of ras and protein kinase C [53], while CD154 gene transcription requires a combination of both calcium-dependent and ras/protein kinase C-mediated signals [45] Although the role of CD69 in T-cell activation or effector function remains poorly understood, CD154 expression has previously been shown to play multiple roles in naive CD4 T-cell activation that are mediated, in large part, by engagement of CD40 on DCs [44] These include increasing DC expression of costimulatory molecules and the production of IL-12 p70 [54] A decrease in these CD154-dependent mechanisms is also likely to account for the absence of antigenspecific memory CD4 T cells secreting IFN-␥ in human beings with genetic deficiency of CD154 [49] Using a neutralizing antibody to CD154, we confirmed that the DC-derived IL-12 p70 production and early IFN-␥ expression by APB naive CD4 T cells was CD154-dependent after allogeneic stimulation with DCs CD154 neutralization also reduced the relatively low levels of production of IL-12 by DCs and IFN-␥ by naive CD4 T cells in CB CD4 T-cell/DC cocultures, indicating a CD154-dependent pathway for Th1 differentiation also applied to CB CD4 T cells in an allogeneic context Studies by others have also demonstrated the importance of CD154 [55] and IL12/STAT4 [9] in the pathogenesis of acute GVHD Together, these results indicate that reduced CD154 expression by CB naive CD4 T cells after allostimulation and its impact on recipient DC cytokine production plays an important role in limiting early Th1 cell differentiation of cells involved in acute GVHD We found that allogeneic priming of CB or APB naive CD4 T cells resulted in increased STAT4 mRNA and protein expression To our knowledge, this striking up-regulation of STAT4 protein expression by T-cell activation has not been described previously, and determining the signal transduction and molecular mechanisms by which this is achieved will be of interest Our data confirm a previous report of decreased STAT4 transcript levels in CB T cells by Impaired Neonatal CD4 T-Cell Allogeneic Activation microarray analysis [56] We furthered these findings by showing that CB naive CD4 T cells had significantly lower basal levels of STAT4 protein than APB naive CD4 T cells, and a relatively reduced level of STAT4 protein by CB CD4 T cells persisted after allogeneic priming This reduced expression of STAT4 may have contributed to the decreased frequency of IL-12-induced pY-STAT4 in naive CB CD4 T cells compared with APB cells after allogeneic priming These results not exclude the possibility that allogeneic priming was less effective in CB naive CD4 T cells in up-regulating IL-12 receptor surface expression, although our inability to flow cytometrically detect the two components of the IL-12 receptor, IL-12R1 or IL-122, on allogeneically primed CD4 T cells precluded testing this possibility Regardless of the relative contribution of receptor versus postreceptor limitations in IL-12 signaling, our results suggest that not only decreased IL-12 production but also reduced STAT4-mediated events limit the Th1 differentiation of naive CB CD4 T cells by alloantigen Because STAT4 appears to play an important role in acute GVHD [9], both reduced IL-12 production and a diminished capacity to mediate STAT4-dependent events may contribute to the reduced risk of acute GVHD after transplantation with allogeneic CB IFN-␥ appears to up-regulate expression of T-bet, a master transcription factor that promotes Th1 differentiation, although the precise mechanism by which T-bet acts remains controversial IFN-␥ may also act directly on activated naive CD4 T cells to maintain IL-12R2 expression, and this effect is dependent on STAT1 [20] Therefore, it is plausible that reduced early IFN-␥ secretion by naive CB CD4 T cells after allogeneic stimulation might act in an autocrine or paracrine manner to reduce Th1 differentiation compared with ABP CD4 T cells IL-10 is an anti-inflammatory cytokine known to decrease tumor necrosis factor-␣ and IFN-␥ production and aid in graft tolerance, possibly through the production of regulatory T-cell populations [57] In addition, IL-10 production by donor T cells has been shown to reduce the severity of acute GVHD in animal models [58] Moreover, others have demonstrated increased levels of IL-10 from CB naive CD4 T cells that have been stimulated with anti-CD3 mAb with or without anti-CD28 mAb and IL-2 for days [59] or after phorbol myristate acetate and ionomycin stimulation after a 7-day co-culture with mature monocytederived DCs [35] These observations raised the possibility that regulatory T cells may be increased in the CB population based on this cytokine profile However, we found that co-cultures of DCs with naive CD4 T cells derived from either CB or APB produced similar low but detectable amounts of IL-10 after 48 hours of co-culture It is possible that IL-10 produc- BB&MT tion differs based on the mode of stimulation (DCs versus anti-CD3 mAb versus phorbol ester/ionomycin), or that increased incubation time is necessary for optimum IL-10 expression, and that IL-10 may play a role in inhibiting later events in acute GVHD Regardless of the cellular source, the higher IL10:IFN-␥ ratio for CB compared with the APB naive CD4 T-cell co-cultures with DCs, which is a result of the reduced secretion of IFN-␥ in the CB co-cultures, might limit IFN-␥-dependent functions For example, IL-10 may reduce IFN-␥-mediated gene expression by suppressing STAT1 tyrosine phosphorylation [60], it is plausible that this high ratio might limit IFN-␥dependent enhancement of Th1 differentiation, such as augmented IL-12 receptor expression, which also requires STAT1 [20] We considered the possibility that decreased priming of CB naive CD4 T cells toward Th1 responses might be a result of increased proportion of regulatory T cells in this naive cell population compared with that of ABP Although our data support prior observations that CB and APB have similar numbers of naive CD4 T cells that are CD25high [26], the observation that a smaller fraction of these express FoxP3 protein suggests that the naive CD4 T-cell compartment of CB has a lower concentration of bona fide Tregs than naive CD4 T cells of APB Together, these results suggest that the decreased Th1 differentiation observed after allogeneic stimulation of naive CD4 T cells from CB is unlikely to be a result of increased Treg activity in this cell population, but rather defects that are intrinsic to naive CD4 T cells that are CD25Ϫ/low The results of these studies are also of interest in defining limitations in CB CD4 T-cell immunity, particularly by Th1 cells, in response to infection or vaccination For example, the human neonate and infant are highly vulnerable to severe or persistent infection with herpesviruses, such as herpes simplex virus and cytomegalovirus, and these susceptibilities are associated with decreased and delayed acquisition of antigen-specific CD4 T-cell immunity, including Th1 function, compared with adults [41,61] Because recent studies support the idea that ␣-TCR engagement by complexes of either peptide/allogeneic MHC or foreign peptide/self-MHC are structurally similar [62], it is likely that these limitations of CD4 T-cell activation by allogeneic stimulation also apply to activation by foreign peptides bound to self-MHC molecules, and contribute to the reduced CD4 T-cell responses of the neonate to intracellular infections, such as with herpesviruses [39] In summary, our results reveal multiple mechanisms–including reduced expression of IFN-␥, IL-2, IL-12, CD154, and a reduced capacity for IL-12dependent STAT4 tyrosine phosphorylation–that limit the activation of CB naive CD4 T cells and their 169 L Chen et al Th1 polarization after allostimulation by mature DCs These in vitro studies argue strongly for a functional limitation in naive CB T-cell activation in vivo in response to allogeneic stimuli as an important mechanism for the relatively low risk of acute GVHD after transplantation using allogeneic CB 13 14 ACKNOWLEDGMENTS We are grateful to El Camino Hospital, Los Altos, Calif, and Pamela Stepick-Biek for help in obtaining umbilical vein CB samples; Dr Seth Lederman (Columbia University) for the 5c8 mAb; and Dr Martin E Dahl for critique of the manuscript This work was supported by a Walter V and Idun Y Berry Fellowship in Children’s Health (A.C.C.), a National Institutes of Health grant K08 AI057961-01 (A.C.C.), an American Society of Hematology Fellow Basic Science Award (A.C.C.), and the Jeffrey Modell Center for Primary Immunodeficiency (D.B.L.) REFERENCES Ferrara JL, Cooke KR, Teshima T The pathophysiology of acute graft-versus-host disease Int J Hematol 2003;78:181-187 Devetten MP, Vose JM Graft-versus-host disease: how to translate new insights into new therapeutic strategies Biol Blood Marrow Transplant 2004;10:815-825 Wysocki CA, Panoskaltsis-Mortari A, Blazar BR, et al Leukocyte migration and graft-versus-host disease Blood 2005;105: 4191-4199 Duffner UA, Maeda Y, Cooke KR, et al Host dendritic cells alone are sufficient to initiate acute graft-versus-host disease J Immunol 2004;172:7393-7398 Anderson BE, McNiff J, Yan J, et al Memory CD4ϩ T cells not induce graft-versus-host disease J Clin Invest 2003;112: 101-108 Foster AE, Marangolo M, Sartor MM, et al Human CD62Lmemory T cells are less responsive to alloantigen stimulation than CD62Lϩ naive T cells: potential for adoptive immunotherapy and allodepletion Blood 2004;104:2403-2409 Beilhack A, Schulz S, Baker J, et al In vivo analyses of early events in acute graft-versus-host disease reveal sequential infiltration of T-cell subsets Blood 2005;106:1113-1122 Teshima T, Ordemann R, Reddy P, et al Acute graft-versushost disease does not require alloantigen expression on host epithelium Nat Med 2002;8:575-581 Nikolic B, Lee S, Bronson RT, et al Th1 and Th2 mediate acute graft-versus-host disease, each with distinct end-organ targets J Clin Invest 2000;105:1289-1298 10 Rocha V, Labopin M, Sanz G, et al Transplants of umbilicalcord blood or bone marrow from unrelated donors in adults with acute leukemia N Engl J Med 2004;351:2276-2285 11 Laughlin MJ, Eapen M, Rubinstein P, et al Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukemia N Engl J Med 2004;351:22652275 12 de Vries E, de Bruin-Versteeg S, Comans-Bitter WM, et al 170 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Longitudinal survey of lymphocyte subpopulations in the first year of life Pediatr Res 2000;47:528-537 Zeng D, Hoffmann P, Lan F, et al Unique patterns of surface receptors, cytokine secretion, and immune functions distinguish T cells in the bone marrow from those in the periphery: impact on allogeneic bone marrow transplantation Blood 2002; 99:1449-1457 Casanova JL, Abel L Genetic dissection of immunity to mycobacteria: the human model Annu Rev Immunol 2002;20:581620 Nel AE T-cell activation through the antigen receptor Part 1: signaling components, signaling pathways, and signal integration at the T-cell antigen receptor synapse J Allergy Clin Immunol 2002;109:758-770 Mellman I, Steinman RM Dendritic cells: specialized and regulated antigen processing machines Cell 2001;106:255-258 Trinchieri G Interleukin-12 and the regulation of innate resistance and adaptive immunity Nat Rev Immunol 2003;3:133146 Mullen AC, High FA, Hutchins AS, et al Role of T-bet in commitment of TH1 cells before IL-12-dependent selection Science 2001;292:1907-1910 Watford WT, Hissong BD, Bream JH, et al Signaling by IL-12 and IL-23 and the immunoregulatory roles of STAT4 Immunol Rev 2004;202:139-156 Berenson LS, Ota N, Murphy KM Issues in T-helper development–resolved and unresolved Immunol Rev 2004;202:157174 Szabo SJ, Sullivan BM, Peng SL, et al Molecular mechanisms regulating Th1 immune responses Annu Rev Immunol 2003; 21:713-758 Schwartz RH Natural regulatory T cells and self-tolerance Nat Immunol 2005;6:327-330 Belkaid Y, Rouse BT Natural regulatory T cells in infectious disease Nat Immunol 2005;6:353-360 Fontenot JD, Gavin MA, Rudensky AY Foxp3 programs the development and function of CD4ϩCD25ϩ regulatory T cells Nat Immunol 2003;4:330-336 Hori S, Nomura T, Sakaguchi S Control of regulatory T cell development by the transcription factor Foxp3 Science 2003; 299:1057-1061 Takahata Y, Nomura A, Takada H, et al CD25ϩCD4ϩ T cells in human cord blood: an immunoregulatory subset with naive phenotype and specific expression of forkhead box p3 (Foxp3) gene Exp Hematol 2004;32:622-629 Wing K, Larsson P, Sandstrom K, et al CD4ϩ CD25ϩ FOXP3ϩ regulatory T cells from human thymus and cord blood suppress antigen-specific T cell responses Immunology 2005;115:516525 Godfrey WR, Spoden DJ, Ge YG, et al Cord blood CD4(ϩ)CD25(ϩ)-derived T regulatory cell lines express FoxP3 protein and manifest potent suppressor function Blood 2005; 105:750-758 Wing K, Lindgren S, Kollberg G, et al CD4 T cell activation by myelin oligodendrocyte glycoprotein is suppressed by adult but not cord blood CD25ϩ T cells Eur J Immunol 2003;33: 579-587 Durandy A, De-Saint-Basile G, Lisowska GB, et al Undetectable CD40 ligand expression on T cells and low B cell responses to CD40 binding agonists in human newborns J Immunol 1995;154:1560-1568 Fuleihan R, Ahern D, Geha RS Decreased expression of the Impaired Neonatal CD4 T-Cell Allogeneic Activation 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 ligand for CD40 in newborn lymphocytes Eur J Immunol 1994;24:1925-1928 Nonoyama S, Penix LA, Edwards CP, et al Diminished expression of CD40 ligand by activated neonatal T cells J Clin Invest 1995;95:66-75 Jullien P, Cron RQ, Dabbagh K, et al Decreased CD154 expression by neonatal CD4ϩ T cells is due to limitations in both proximal and distal events of T cell activation Int Immunol 2003;15:1461-1472 Matthews NC, Wadhwa M, Bird C, et al Sustained expression of CD154 (CD40L) and proinflammatory cytokine production by alloantigen-stimulated umbilical cord blood T cells J Immunol 2000;164:6206-6212 Liu E, Law HKW, Lau YL Tolerance associated with cord blood transplantation may depend on the state of the host dendritic cells Br J Hematol 2004;126:517-526 Splawski JB, Nishioka J, Nishioka Y, et al CD40 ligand is expressed and functional on activated neonatal T cells J Immunol 1996;156:119-127 Lee SM, Suen Y, Chang L, et al Decreased interleukin-12 (IL-12) from activated cord versus adult peripheral blood mononuclear cells and upregulation of interferon-gamma, natural killer, and lymphokine-activated killer activity by IL-12 in cord blood mononuclear cells Blood 1996;88:945-954 Early E, Reen DJ Rapid conversion of naive to effector T cell function counteracts diminished primary human newborn T cell responses Clin Exp Immunol 1999;116:527-533 Lewis DB, Wilson CB Developmental immunology and role of host defenses in the fetal and neonatal susceptibility to infection In: Remington JS, Klein JO, Wilson CB, eds Infectious Diseases of the Fetus and Newborn Infant 6th ed Philadelphia, PA, Elsevier 2005:87-210 Cleary AM, Tu W, Enright A, et al Impaired accumulation and function of memory CD4 T cells in human IL-12 receptor beta deficiency J Immunol 2003;170:597-603 Tu W, Chen S, Sharp M, et al Persistent and selective deficiency of CD4ϩ T cell immunity to cytomegalovirus in immunocompetent young children J Immunol 2004;172:3260-3267 Uzel G, Frucht DM, Fleisher TA, et al Detection of intracellular phosphorylated STAT-4 by flow cytometry Clin Immunol 2001;100:270-276 Ziegler SF, Ramsdell F, Alderson MR The activation antigen CD69 Stem Cells 1994;12:456-465 Quezada SA, Jarvinen LZ, Lind EF, et al CD40/CD154 interactions at the interface of tolerance and immunity Annu Rev Immunol 2004;22:307-328 Crabtree GR, Olson EN NFAT signaling: choreographing the social lives of cells Cell 2002;109(suppl):S67-S79 Frucht DM, Fukao T, Bogdan C, et al IFN-gamma production by antigen-presenting cells: mechanisms emerge Trends Immunol 2001;22:556-560 Delespesse G, Yang LP, Ohshima Y, et al Maturation of human neonatal CD4ϩ and CD8ϩ T lymphocytes into Th1/ Th2 effectors Vaccine 1998;16:1415-1419 BB&MT 48 Moore KW, de Waal Malefyt R, Coffman RL, et al Interleukin-10 and the interleukin-10 receptor Annu Rev Immunol 2001;19:683-765 49 Ameratunga R, Lederman HM, Sullivan KE, et al Defective antigen-induced lymphocyte proliferation in the X-linked hyper-IgM syndrome [see comments] J Pediatr 1997;131:147150 50 Garderet L, Dulphy N, Douay C, et al The umbilical cord blood alpha/beta T-cell repertoire: characteristics of a polyclonal and naive but completely formed repertoire Blood 1998; 91:340-346 51 Chandok MR, Farber DL Signaling control of memory T cell generation and function Semin Immunol 2004;16:285-293 52 Castellanos MC, Munoz C, Montoya MC, et al Expression of the leukocyte early activation antigen CD69 is regulated by the transcription factor AP-1 J Immunol 1997;159:5463-5473 53 Zhong XP, Hainey EA, Olenchock BA, et al Regulation of T cell receptor-induced activation of the Ras-ERK pathway by diacylglycerol kinase zeta J Biol Chem 2002;277:31089-31098 54 Cella M, Scheidegger D, Palmer-Lehmann K, et al Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation J Exp Med 1996;184:747-752 55 Buhlmann JE, Gonzalez M, Ginther B, et al Cutting edge: sustained expansion of CD8ϩ T cells requires CD154 expression by Th cells in acute graft versus host disease J Immunol 1999;162:4373-4376 56 Kaminski BA, Kadereit S, Miller RE, et al Reduced expression of NFAT-associated genes in UCB versus adult CD4ϩ T lymphocytes during primary stimulation Blood 2003;102:46084617 57 Groux H, O’Garra A, Bigler M, et al A CD4ϩ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis Nature 1997;389:737-742 58 Blazar BR, Taylor PA, Panoskaltsis-Mortari A, et al Interleukin-10 dose-dependent regulation of CD4ϩ and CD8ϩ T cellmediated graft-versus-host disease Transplantation 1998;66: 1220-1229 59 Rainsford E, Reen DJ Interleukin 10, produced in abundance by human newborn T cells, may be the regulator of increased tolerance associated with cord blood stem cell transplantation Br J Haematol 2002;116:702-709 60 Ito S, Ansari P, Sakatsume M, et al Interleukin-10 inhibits expression of both interferon alpha- and interferon gammainduced genes by suppressing tyrosine phosphorylation of STAT1 Blood 1999;93:1456-1463 61 Burchett SK, Corey L, Mohan KM, et al Diminished interferongamma and lymphocyte proliferation in neonatal and postpartum primary herpes simplex virus infection J Infect Dis 1992; 165:813-818 62 Housset D, Malissen B What TCR-pMHC crystal structures teach us about MHC restriction and alloreactivity? Trends Immunol 2003;24:429-437 171